Hyaluronic acid

HCC tumor tissues were harvested, minced, and digested with 0.1 mg/ml DNAse (Roche), 1 mg/ml type IV collagenase (Invitrogen) and 0.5% hyaluronic acid (Solarbio) for 1 h at 37 °C. The homogenates were passed through 200-mesh stainless steel strainers to obtain single cell suspension. The tumor cells were pelleted with 700 rpm for 1 min at the bottom of the centrifuge tube. For qRT-PCR analysis, total RNAs were extracted with TRIzol. For flow cytometry analysis, cells were fixed with 4% paraformaldehyde and 0.5% TritonX-100 (Sigma), blocked with 0.1% rat serum in PBS and stained with fluorescence-conjugated anti-Ki67 antibody (Clone 16A8, Biolegend, San Diego, CA, USA) for 40 min at 4°C. The stained cells were acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Hepatocellular carcinoma mouse liver and tumour tissues were harvested and disaggregated by mechanic mincing and enzymatic digestion with 0.1 mg·mL^–1 DNAse (Roche), 1 mg·mL^–1 type IV collagenase (Invitrogen), and 0.5% hyaluronic acid (Solarbio) for 1 h at 37 °C, then squeezed through a 200‐mesh stainless steel strainer, and the cell suspension was collected. After a brief centrifugation at 100g for 1 min, the liver or tumour cells were at the bottom of tube, and mononuclear cells were isolated from the upper suspensions using Percoll gradient centrifugation as described before [25 (link)]. After washing with PBS, all types of cells were used for flow cytometry analysis.